The long-term objective of the applicant's laboratory is to improve chemotherapy with camptothecins (CPT) and other topoisomerase I-directed agents. The anti-cancer activity of CPT is determined by factors that include the cellular quantity of topoisomerase I (TOP1), its localization to nuclear sites where it can be converted into lethal damage, and the repair of the TOP1-DNA covalent complex. Posttranslational modifications are implicated to regulate each of these factors, but these regulatory pathways are poorly understood. Knowledge of these pathways can be applicable for predicting therapeutically responsive tumor subtypes and for designing new chemotherapeutic targets for use in combination with TOP1-targeting chemotherapeutics. The objective of this application is to identify post-translational regulatory mechanisms of TOP1, and to determine relationships among post-translational modifications, TOP1 localization and accumulation, and effectiveness of TOP1 as a drug target. The Specific Aims are to: 1) Identify sites of phosphorylation on TOP1 and phosphorylation sites, whose phosphorylation state changes during CPT treatment. Post-translational modification sites will be identified using a combination of site directed mutagenesis, phosphopeptide mapping, and mass spectrometry. 2) Determine the regulatory role of TOP1 modifications. Mutations will be used to abolish and mimic post-translational modifications of TOP1, and the effect of the modification-site mutations on the regulation of TOP1 will be determined. Modification-state specific antibodies will also be prepared for studies of the post-translational regulation of endogenous TOP1. 3) Determine the effect of BTBD1 and BTBD2 on the cullin-3 (Cul3)-promoted downregulation of the TOP1-DNA covalent complex. BTBD1 and BTBD2 are putative substrate receptors for Cul3 and will be tested with regard to their role in the Cul3-promoted downregulation of TOP1. 4) Determine the extent to which post-translational modifications of TOP1 modulate its effectiveness as a drug target. Phosphorylation-site mutant TOP1 will be compared with normal TOP1 with regard to their effectiveness for mediating CPT-induced cell killing. This research is expected to ultimately impact on chemotherapy with TOP1-targeting agents by leading to the identification of predictors of therapeutic response, new drug targets, and synergistic co-therapies with TOP1-targeting chemotherapeutics.